This invention is in the field of medicinal chemistry and relates to novel compounds, and pharmaceutical compositions thereof, that inhibit caspases that mediate cell apoptosis and inflammation. The invention also relates to methods of using the compounds and pharmaceutical compositions of this invention to treat diseases where caspase activity is implicated.
Apoptosis, or programmed cell death, is a principal mechanism by which organisms eliminate unwanted cells. The deregulation of apoptosis, either excessive apoptosis or the failure to undergo it, has been implicated in a number of diseases such as cancer, acute inflammatory and autoimmune disorders, ischemic diseases and certain neurodegenerative disorders (see generally Science, 1998, 281, 1283-1312; Ellis et al., Ann. Rev. Cell. Biol., 1991, 7, 663).
Caspases are a family of cysteine protease enzymes that are key mediators in the signaling pathways for apoptosis and cell disassembly (Thornberry, Chem. Biol., 1998, 5, R97-R103). These signaling pathways vary depending on cell type and stimulus, but all apoptosis pathways appear to converge at a common effector pathway leading to proteolysis of key proteins. Caspases are involved in both the effector phase of the signaling pathway and further upstream at its initiation. The upstream caspases involved in initiation events become activated and in turn activate other caspases that are involved in the later phases of apoptosis.
Caspase-1, the first identified caspase, is also known as interleukin converting enzyme or xe2x80x9cICE.xe2x80x9d Caspase-1 converts precursor interleukin-1xcex2 (xe2x80x9cpIL-1xcex2xe2x80x9d) to the pro-inflammatory active form by specific cleavage of pIL-1xcex2 between Asp-116 and Ala-117. Besides caspase-1 there are also eleven other known human caspases, all of which cleave specifically at aspartyl residues. They are also observed to have stringent requirements for at least four amino acid residues on the N-terminal side of the cleavage site.
The caspases have been classified into three groups depending on the amino acid sequence that is preferred or primarily recognized. The group of caspases, which includes caspases 1, 4, and 5, has been shown to prefer hydrophobic aromatic amino acids at position 4 on the N-terminal side of the cleavage site. Another group which includes caspases 2, 3 and 7, recognize aspartyl residues at both positions 1 and 4 on the N-terminal side of the cleavage site, and preferably a sequence of Asp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10, tolerate many amino acids in the primary recognition sequence, but seem to prefer residues with branched, aliphatic side chains such as valine and leucine at position 4.
The caspases have also been grouped according to their perceived function. The first subfamily consists of caspases-1 (ICE), 4, and 5. These caspases have been shown to be involved in pro-inflammatory cytokine processing and therefore play an important role in inflammation. Caspase-1, the most studied enzyme of this class, activates the IL-1xcex2 precursor by proteolytic cleavage. This enzyme therefore plays a key role in the inflammatory response. Caspase-1 is also involved in the processing of interferon gamma inducing factor (IGIF or IL-18) which stimulates the production of interferon gamma, a key immunoregulator that modulates antigen presentation, T-cell activation and cell adhesion.
The remaining caspases make up the second and third subfamilies. These enzymes are of central importance in the intracellular signaling pathways leading to apoptosis. One subfamily consists of the enzymes involved in initiating events in the apoptotic pathway, including transduction of signals from the plasma membrane. Members of this subfamily include caspases-2, 8, 9 and 10. The other subfamily, consisting of the effector capsases 3, 6 and 7, are involved in the final downstream cleavage events that result in the systematic breakdown and death of the cell by apoptosis. Caspases involved in the upstream signal transduction activate the downstream caspases, which then disable DNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton and finally fragment the cell.
Knowledge of the four amino acid sequence primarily recognized by the caspases has been used to design caspase inhibitors. Reversible tetrapeptide inhibitors have been prepared having the structure CH3CO-[P4]-[P3]-[P2]-CH(R)CH2CO2H where P2 to P4 represent an optimal amino acid recognition sequence and R is an aldehyde, nitrile or ketone capable of binding to the caspase cysteine sulfhydryl. Rano and Thornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med. Chem. Lett. 3, 2689-2692 (1993); Nicholson et al., Nature 376, 37-43 (1995). Irreversible inhibitors based on the analogous tetrapeptide recognition sequence have been prepared where R is an acyloxymethylketone xe2x80x94COCH2OCORxe2x80x2. Rxe2x80x2 is exemplified by an optionally substituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH2X where X is a leaving group such as F or Cl. Thornberry et al., Biochemistry 33, 3934 (1994); Dolle et al., J Med. Chem. 37, 563-564 (1994).
The utility of caspase inhibitors to treat a variety of mammalian diseases associated with an increase in cellular apoptosis has been demonstrated using peptidic caspase inhibitors. For example, in rodent models, caspase inhibitors have been shown to reduce infarct size and inhibit cardiomyocyte apoptosis after myocardial infarction, to reduce lesion volume and neurological deficit resulting from stroke, to reduce post-traumatic apoptosis and neurological deficit in traumatic brain injury, to be effective in treating fulminant liver destruction, and to improve survival after endotoxic shock. Yaoita et al., Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow and Metabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992 (1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez et al., J. Exp. Med., 184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585 (1999).
In general, the peptidic inhibitors described above are very potent against some of the caspase enzymes. However, this potency has not always been reflected in cellular models of apoptosis. In addition peptide inhibitors are typically characterized by undesirable pharmacological properties such as poor oral absorption, poor stability and rapid metabolism. Plattner and Norbeck, in Drug Discovery Technologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England, 1990).
Recognizing the need to improve the pharmacological properties of the peptidic caspase inhibitors, peptidomimetic and non-natural amino acid peptide inhibitors have been reported.
WO 96/40647 discloses ICE inhibitors of the formula: 
wherein B is H or an N-terminal blocking group; R1 is the amino acid side chain of the P1 amino acid residue wherein the P1 amino acid is not Asp; Pn is an amino acid residue or a heterocyclic replacement of the amino acid; R4 is hydroxyl, alkoxyl, acyl, hydrogen, alkyl or phenyl; m is 0 or a positive integer; and X is N, S, O, or CH2.
U.S. Pat. No. 5,585,357 discloses compounds which inhibit interleukin-1xcex2 protease. These inhibitors are represented by the formula: 
wherein each AA is independently L-valine or L-alanine; n is 0-2; R1 is certain groups; and R8, R9, R10 are each independently hydrogen, lower alkyl, halo substituted methyl, carbalkoxy, benzyl, phenyl or phenyl mono- or disubstituted with fluoro, nitro, methoxy, chloro, trifluoromethyl or methanesulfonyl.
WO 98/16502 discloses aspartate ester inhibitors of interleukin-1xcex2 converting enzyme of the formula: 
wherein R1 is, inter alia, R5N(Ra)CHR6COxe2x80x94; R2 is certain groups; R6 is H, C1-6 alkyl, xe2x80x94(CH2)n, aryl, xe2x80x94(CH2)nCO2Ra, hydroxyl substituted C1-6 alkyl, or imidazole substituted C1-6 alkyl; and Ra is independently hydrogen, C1-6 alkyl or xe2x80x94(CH2)naryl
WO 99/18781 discloses dipeptide apoptosis inhibitors having the formula: 
where R1 is an N-terminal protecting group; AA is a residue of any natural xcex1-amino acid, or xcex2-amino acid; and R2 and R3 are defined in the application.
WO 00/023421 discloses (substituted)acyl dipeptide apoptosis inhibitors having the formula: 
wherein n is 0, 1, or 2; q is 1 or 2; A is a residue of any natural or non-natural amino acid; B is a hydrogen atom, a deuterium atom, C1-10 straight chain or branched alkyl, cycloalkyl, phenyl, substituted phentyl, naphthyl, substituted naphthyl, 2-benzoxazolyl, substituted 2-oxazolyl, (CH2)mcycloalkyl, (CH2)mphenyl, (CH2)m(substituted phenyl), (CH2)m(1- or 2-naphthyl), (CH2)mheteroaryl, halomethyl, CO2R13, CONR14R15, CH2ZR16, CH2OCOaryl, CH2OCO(substituted aryl), CH2OCO(heteroaryl), CH2OCO(substituted heteroaryl), or CH2OPO(R17)R18, where R13, R14, R15, R16, R17 and R18 are defined in the application; R2 is selected from a group containing hydrogen, alkyl, cycloalkyl, phenyl, substituted phenyl, (CH2)mNH2; R3 is hydrogen, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or substituted phenylalkyl; X is CH2, Cxe2x95x90O, O, S, NH, Cxe2x95x90ONH or CH2OCONH; and Z is an oxygen or a sulfur atom.
WO 00/061542 discloses dipeptide apoptosis inhibitors having the formula: 
where R1 is an optionally substituted alkyl or hydrogen group; R2 is hydrogen or optionally substituted alkyl; Y is a residue of a natural or non-natural amino acid and R3 is an alkyl, saturated carbocyclic, partially saturated carbocyclic, aryl, saturated heterocyclic, partially saturated heterocyclic or heteroaryl group, wherein said group is optionally substituted; X is O, S, NR4, or (CR4R5)n where R4 and R5 are, at each occurrence, independently selected from the group consisting of hydrogen, alkyl and cycloalkyl, and n is 0, 1, 2, or 3; or X is NR4, and R3 and R4 are taken together with the nitrogen atom to which they are attached to form a saturated heterocyclic, partially saturated heterocyclic or heteroaryl group, wherein said group is optionally substituted or X is CR4R5, and R3 and R4 are taken together with the carbon atom to which they are attached to form a saturated carbocyclic, partially saturated carbocyclic, aryl, saturated heterocyclic, partially saturated heterocyclic or oxygen-containing heteroaryl group, wherein said group is optionally substituted; and provided that when X is O, then R3 is not unsubstituted benzyl or t-butyl; and when X is CH2, then R3 is not H.
While a number of caspase inhibitors have been reported, it is not clear whether they possess the appropriate pharmacological properties to be therapeutically useful. Therefore, there is a continued need for small molecule caspase inhibitors that are potent, stable, and penetrate membranes to provide effective inhibition of apoptosis in vivo. Such compounds would be extremely useful in treating the aforementioned diseases where caspase enzymes play a role.
It has now been found that compounds of this invention and pharmaceutical compositions thereof are particularly effective as inhibitors of caspases and cellular apoptosis. These compounds have the general formula I: 
wherein:
Ring A is an optionally substituted piperidine, tetrahydroquinoline or tetrahydroisoquinoline ring;
R1 is hydrogen, CN, CHN2, R, or CH2Y;
R is an optionally substituted group selected from an aliphatic group, an aryl group, or an aralkyl group;
Y is an electronegative leaving group;
R2 is CO2H, CH2CO2H, or esters, amides or isosteres thereof; and
R3 is hydrogen, an optionally substituted aryl group, an optionally substituted aralkyl group, or an optionally substituted C1-6 aliphatic group, R4 is an optionally substituted group selected from an aryl group or a heterocyclyl group, or R3 and R4 taken together with the nitrogen to which they are attached optionally form is a substituted or unsubstituted monocyclic, bicyclic or, tricyclic ring.
The compounds of this invention have potent inhibition properties across a range of caspase targets with good efficacy in cellular models of apoptosis. In addition, these compounds are expected to have improved cell penetration and pharmacokinetic properties and, as a consequence of their potency, have improved efficacy against diseases where caspases are implicated.